Polyethylene (Glycol) derivatives with proximal reactive groups

ABSTRACT

An activated, substantially water soluble poly(ethylene glycol) is provided having of a linear or branched poly(ethylene glycol) backbone and at least one terminus linked to the backbone through a hydrolytically stable linkage, wherein the terminus is branched and has proximal reactive groups. The free reactive groups are capable of reacting with active moieties in a biologically active agent such as a protein or peptide thus forming conjugates between the activated poly(ethylene glycol) and the biologically active agent.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a divisional of U.S. application Ser. No.09/265,989, now U.S. Pat. No. ______, filed on Mar. 11, 1999, which isrelated to Provisional Application Serial No. 60/077,700, filed Mar. 12,1998, and claims the benefit of its filing date under 35 U.S.C. §119(e).Both applications are incorporated by reference herein in theirentirety.

FIELD OF THE INVENTION

[0002] This invention relates to derivatives of polyethylene glycol andrelated hydrophilic polymers suitable for chemical coupling to anothermolecule, including, for example, proteins, enzymes, small drugs, andthe like.

BACKGROUND OF THE INVENTION

[0003] Chemical attachment of the hydrophilic polymer poly(ethyleneglycol) (“PEG”) to molecules and surfaces is of great utility inbiotechnology. In its most common form PEG is a linear polymerterminated at each end with hydroxyl groups:

HO—CH₂CH₂O—(CH₂CH₂O)_(n)—CH₂CH₂—OH

[0004] This polymer can be represented in brief form as HO—PEG-OH whereit is understood that the —PEG- symbol represents the followingstructural unit:

—CH₂CH₂O—(CH₂CH₂O)_(n)—CH₂CH₂—

[0005] In typical form n ranges from about 10 to about 2000.

[0006] PEG is commonly used as methoxy PEG-OH, or MPEG in brief, inwhich one terminus is the relatively inert methoxy group, while theother terminus is a hydroxyl group that is subject to ready chemicalmodification.

CH₃O—(CH₂CH₂O)_(n)—CH₂CH₂—OH MPEG

[0007] PEG is also commonly used in branched forms that can be preparedby addition of ethylene oxide to various polyols, such as glycerol,pentaerythritol and sorbitol. For example, the four-arm, branched PEGprepared from pentaerythritol is shown below:

C(CH₂—OH)₄+n C₂H₄O→C[CH₂—O—(CH₂CH₂O)_(n)—CH₂CH₂—OH]₄

[0008] The branched PEGs can be represented in general form asR(—PEG-OH)_(n) in which R represents the central “core” molecule, suchas glycerol or pentaerythritol, and n represents the number of arms.

[0009] Branched PEGs can also be prepared in which two PEG “arms” areattached to a central linking moiety having a single functional groupcapable of joining to other molecules; e.g., Matsushima et al., (Chem.Lett., 773, 1980) have coupled two PEGs to a central cyanuric chloridemoiety.

[0010] PEG is a well known polymer having the properties of solubilityin water and in many organic solvents, lack of toxicity, and lack ofimmunogenicity. One use of PEG is to covalently attach the polymer toinsoluble molecules to make the resulting PEG-molecule “conjugate”soluble. For example, it has been shown that the water-insoluble drugpaclitaxel, when coupled to PEG, becomes water-soluble. Greenwald, etal., J. Org. Chem., 60:331-336 (1995).

[0011] In related work, U.S. Pat. No. 4,179,337 to Davis et al.discloses that proteins coupled to PEG have enhanced blood circulationlifetime because of reduced rate of kidney clearance and reducedimmunogenicity. These and other applications are also described inBiomedical and Biotechnical Applications of Polyethylene GlycolChemistry, J. M. Harris, Ed., Plenum, N.Y. (1992), and Poly(ethyleneglycol) Chemistry and Biological Applications, J. M. Harris and S.Zalipsky, Eds., ACS, Washington D.C. (1997).

[0012] To couple PEG to a molecule such as a protein, it is oftennecessary to “activate” the PEG to prepare a derivative of the PEGhaving a functional group at the terminus. The functional group canreact with certain moieties on the protein such as an amino group, thusforming a PEG-protein conjugate. Many activated derivatives of PEG havebeen described. An example of such an activated derivative is thesuccinimidyl succinate “active ester”:

CH₃O—PEG-O₂C—CH₂CH₂—CO₂—NS

[0013] where NS=

[0014] Hereinafter, the succinimidyl active ester moiety will berepresented as —CO₂—NS in chemical drawings.

[0015] The succinimidyl active ester is a useful compound because itreacts rapidly with amino groups on proteins and other molecules to forman amide linkage (—CO—NH—). For example, U.S. Pat. No. 4,179,337 toDavis et al. describes coupling of this derivative to proteins(represented as PRO—NH₂):

mPEG-O₂CCH₂CH₂CO₂NS+PRO—NH₂→mPEG-O₂C—CH₂CH₂—CONH—PRO

[0016] Bifunctional PEGs with active groups at both ends of the linearpolymer chain are also useful compounds when formation of a crosslinkedinsoluble network is desired. Many such bifunctional PEGs are known inthe art. For example, U.S. Pat. No. 5,162,430 to Rhee, et al. disclosesusing such bifunctional PEGs to crosslink collagen.

[0017] Reactive PEGs have also been synthesized in which several activefunctional groups are placed along the backbone of the polymer. Forexample, lysine-PEG conjugates have been prepared in the art in which anumber of activated groups are placed along the backbone of the polymer.Zalipsky et al. Bioconjugate Chemistry, 4:54-62 (1993).

[0018] U.S. Pat. No. 5,283,339 to Arnold et al. discloses PEG compoundscapable of chelating metals. The PEG compounds have a terminal metalchelating group which has two free carboxylic acid or amino groups,typically linked to a nitrogen atom. The PEG compounds are used toextract and precipitate proteins from solutions with the carboxylic acidor amino groups together with the nitrogen atom capable of forming ioniccomplexes with metal ions. However, the metal chelating groups disclosedin the patent generally are not useful in covalently coupling the PEGcompounds to proteins, peptides, or small drugs bearing functionalgroups such as amines. The patent does not teach forming an activatedPEG derivative for covalently coupling to another molecule to form aconjugate.

SUMMARY OF THE INVENTION

[0019] The invention described herein provides a water soluble polymersuch as poly(ethylene glycol) or related polymers that have a branchedmoiety at one end of the polymer chain and two free reactive groupslinked to the branched moiety for covalent attachment to anothermolecule. Each reactive moiety can have a tethering group, such as analkyl chain, linking a reactive group to the branched moiety. Thus, thebranched terminus allows the activated water soluble polymer of thisinvention to react with two molecules to form conjugates.

[0020] Because a tethering group having a desirable length can beselected in preparing an activated polymer, the two reactive groups canbe held at a predetermined distance apart from each other. The twomolecules conjugated to the activated polymer through the two reactivegroups can also be held at a predetermined distance apart. Accordingly,an activated PEG is provided in accordance with the invention having twofree reactive moieties branching out from one PEG chain at a branchedmoiety. The two free reactive moieties are capable of reacting withbiologically active agents such as proteins, thereby linking theactivated polymer to the biologically active agents.

[0021] In accordance with one embodiment of this invention, an activatedwater soluble polymer is provided having the formula:

[0022] wherein POLY is a water soluble, substantially non-immunogenicpolymer backbone, Y is a hydrolytically stable linkage, X and X′ arereactive groups capable of reacting with a moiety in another moleculesuch as a protein. Typically, the polymer backbone is selected from thegroup consisting of linear and branched poly(ethylene glycol), linearand branched poly(alkylene oxide), linear and branched poly(vinylpyrrolidone), linear and branched poly(vinyl alcohol), linear andbranched polyoxazoline, linear and branched poly(acryloylmorpholine),and derivatives thereof. Preferably, the polymer backbone ispoly(ethylene glycol) or a derivative thereof. The polymer backbone POLYcan have a capping group selected from the group consisting of —OH,alkyls, and —Y—CHXX′ wherein Y, X and X′ are as described above and canbe the same or different on each terminus of the PEG.

[0023] In a preferred embodiment, X and X′ are represented by —W-Z and—W′-Z′ respectively, in which Z and Z′ represent reactive moieties forconjugating the polymer to another molecule. W and W′ representtethering groups comprising a substantially linear chain of atoms, e.g.,alkyl chains, ether chains, ester chains, amide chains, and combinationsthereof. Examples of the reactive moieties include, but are not limitedto, active esters, active carbonates, aldehydes, isocyanates,isothiocyanates, epoxides, alcohols, maleimides, vinylsulfones,hydrazides, dithiopyridines, and iodoacetamides.

[0024] In another embodiment of the activated polymer of this invention,the activated water soluble polymer has the formula:

[0025] wherein

[0026] R is a central branch core;

[0027] POLY is a water soluble substantially non-immunogenic polymer;

[0028] Y is a hydrolytically stable linkage;

[0029] n is from 2 to 200;

[0030] X and X′ are reactive groups capable of reacting with a moiety inanother molecule such as a protein.

[0031] Many central branch core molecules for preparing branched ordendritic PEGs are known and can all be used for R. Typically, R can bea moiety derived from lysine, glycerol, pentaerythritol, or sorbitol.Suitable polymer backbones include, but are not limited to, linear andbranched poly(ethylene glycol), linear and branched poly(alkyleneoxide), linear and branched poly(vinyl pyrrolidone), linear and branchedpoly(vinyl alcohol), linear and branched polyoxazoline, linear andbranched poly(acryloylmorpholine), and derivatives thereof. Preferably,is poly(ethylene glycol) or a derivative thereof is used as the polymerbackbone.

[0032] The reactive groups X and X′ can be reactive moieties directlylinked to the branching moiety —CH. Preferably, X and X′ have atethering group and are represented by —W-Z and —W′-Z′ respectively, inwhich Z and Z′ represent reactive groups for conjugating the polymer toanother molecule. W and W′ represent tethering groups comprising asubstantially linear chain of atoms, e.g., alkyl chains, ether chains,ester chains, amide chains, and combination thereof. Examples of thereactive groups include, but are not limited to, active esters, activecarbonates, aldehydes, isocyanates, isothiocyanates, epoxides, alcohols,maleimides, vinylsulfones, hydrazides, dithiopyridines, andiodoacetamides.

[0033] The activated water soluble polymer can be covalently linked to abiologically active agent to form a conjugate. A suitable biologicallyactive agent can be any of those having a moiety capable of reactingwith at least one of the two reactive groups in the terminus of theactivated polymer. The biologically active agent can have two suchmoieties and each of them can be linked to one of the two reactivegroups. Alternatively, the conjugate can have two biologically activeagents each being linked to one of the two reactive moieties of theactivated polymer. Because activated polymers having different tetheringgroups can be prepared in accordance with this invention, an activatedpolymer can be provided in which the two reactive groups in a terminusof the activated polymer are a desirable distance from each other. Whensuch an activated polymer is conjugated to two biologically active agentmolecules, the two molecules can be held at a desired distance apart.

[0034] Accordingly, the activated PEG can be used with greaterversatility as compared to other PEG derivatives heretofore known in theart to form various conjugates with molecules such as proteins orpeptides. Since PEG molecules conjugated to another molecule can impartwater solubility and reduced immunogenicity to the other molecule, theactivated PEG derivatives of this invention allows greater control andprecision in modifying such characteristics in a conjugate.

[0035] Thus, an activated water soluble polymer having proximal reactivegroups is provided. The polymer backbone has at least one terminushaving two reactive groups. The terminus has a branching moiety and twofree reactive moieties linked to the branching moiety. The branchingmoiety is in turn linked to the polymer backbone through a stablelinkage.

DETAILED DESCRIPTION OF THE INVENTION

[0036] The terms “group,” “functional group,” “moiety,” “active moiety,”“reactive site,” reactive group” and “reactive moiety” are used in theart and herein to refer to distinct, definable portions or units of amolecule. The terms are somewhat synonymous in the chemical arts and areused herein to indicate that the portions of molecules that perform somefunction or activity and are reactive with other portions of molecules.

[0037] The term “linkage” is used herein to refer to groups or bondsthat normally are formed as the result of a chemical reaction andtypically are covalent linkages. Hydrolytically stable linkages meansthat the linkages are substantially stable in water and do not reactwith water at useful pHs, e.g., under physiological conditions for anextended period of time, preferably indefinitely.

[0038] The term “biologically active agent” when used herein means anysubstance which can affect any physical or biochemical properties of abiological organism, including but not limited to viruses, bacteria,fungi, plants, animals, and humans. In particular, as used herein,biologically active agent includes any substance intended for thediagnosis, cure, mitigation, treatment, or prevention of disease inhumans or other animals, or to otherwise enhance physical or mental wellbeing of humans or animals. Examples of biologically active agentsinclude, but are not limited to, organic and inorganic compounds,proteins, peptides, lipids, polysaccharides, nucleotides, DNAs, RNAs,other polymers, and derivatives thereof. Examples of biologically activeagents include antibiotics, fungicides, anti-viral agents,anti-inflammatory agents, anti-tumor agents, cardiovascular agents,anti-anxiety agents, hormones, growth factors, steroidal agents, and thelike. Other examples include, microorganisms such as bacteria and yeastcells, viral particles, plant or animal or human cells, and the like.

[0039] The polymer backbone is a water soluble substantiallynon-immunogenic polymer, and is preferably poly(ethylene glycol) (PEG).However, it should be understood that other related polymers are alsosuitable for use in the practice of this invention and that the use ofthe term PEG or poly(ethylene glycol) is intended to be inclusive andnot exclusive in this respect.

[0040] Poly(ethylene glycol) or PEG is useful in biological applicationsbecause it has properties that are highly desirable and is generallyapproved for biological or biotechnical applications. PEG typically isclear, colorless, odorless, soluble in water, stable to heat, inert tomany chemical agents, does not hydrolyze or deteriorate, and isgenerally nontoxic. Poly(ethylene glycol) is considered to bebiocompatible, which is to say that PEG is capable of coexistence withliving tissues or organisms without causing harm. More specifically, PEGis non-immunogenic, which is to say that PEG does not tend to produce animmune response in the body. When attached to a molecule having somedesirable function in the body, such as a biologically active agent, toform a conjugate, the PEG tends to mask the agent and can reduce oreliminate any immune response so that an organism can tolerate thepresence of the agent. Accordingly, the conjugate is substantiallynon-toxic. PEG conjugates tend not to produce a substantial immuneresponse or cause clotting or other undesirable effects. PEG having theformula —CH₂CH₂—(CH₂CH₂O)_(n)—CH₂CH₂—, where n is from about 8 to about4000, is one useful polymer in the practice of the invention. PreferablyPEG having a molecular weight of from about 200 to about 100,000 Da isused as polymer backbone.

[0041] The polymer backbone can be linear or branched. Branched polymerbackbones are generally known in the art. Typically, a branched polymerhas a central branch core moiety and a plurality of linear polymerchains linked to the central branch core. PEG is commonly used inbranched forms that can be prepared by addition of ethylene oxide tovarious polyols, such as glycerol, pentaerythritol and sorbitol. Forexample, the four-arm, branched PEG prepared from pentaerythritol isshown below:

C(CH₂—OH)₄+n C₂H₄O→C[CH₂0-(CH₂CH₂O)_(n)—CH₂CH₂—OH]₄

[0042] The central branch moiety can also be derived from several aminoacids, e.g., lysine.

[0043] The branched polyethylene glycols can be represented in generalform as R(—PEG-OH)_(n) in which R represents the core moiety, such asglycerol or pentaerythritol, and n represents the number of arms.Suitable branched PEGs can be prepared in accordance with InternationalPublication No. WO 96/21469, entitled Multi-Armed, Monofunctional, andHydrolytically Stable Derivatives of Poly(Ethylene Glycol)and RelatedPolymers For Modification of Surfaces and Molecules, which was filedJan. 11, 1996, the contents of which are incorporated herein in theirentirety by reference (which corresponds to U.S. Pat. No. 5,932,462,which is also incorporated by reference). These branched PEGs can thenbe modified in accordance with the teachings herein.

[0044] Many other water soluble substantially non-immunogenic polymersthan PEG are also suitable for the present invention. These otherpolymers can be either in linear form or branched form, and include, butare not limited to, other poly(alkylene oxides) such as poly(propyleneglycol) (“PPG”), copolymers of ethylene glycol and propylene glycol andthe like; poly(vinyl alcohol) (“PVA”) and the like. The polymers can behomopolymers or random or block copolymers and terpolymers based on themonomers of the above polymers, straight chain or branched.

[0045] Specific examples of suitable additional polymers include, butare not limited to, difunctional poly(acryloylmorpholine) (“PAcM”), andpoly(vinylpyrrolidone)(“PVP”). PVP and poly(oxazoline) are well knownpolymers in the art and their preparation should be readily apparent tothe skilled artisan. PAcM and its synthesis and use are described inU.S. Pat. Nos. 5,629,384 and 5,631,322, the contents of which areincorporated herein by reference in their entirety.

[0046] Although the molecular weight of each chain of the polymerbackbone can vary, it is typically in the range of from about 100 toabout 100,000, preferably from about 6,000 to about 80,000.

[0047] Those of ordinary skill in the art will recognize that theforegoing list for substantially water soluble non-immunogenic polymerbackbone is by no means exhaustive and is merely illustrative, and thatall polymeric materials having the qualities described above arecontemplated.

[0048] The activated polymer of this invention also has proximalreactive groups linked to at least one arm of the polymer backbone. Aswill be apparent, the term “proximal” is used herein to mean that theterminus has two free reactive moieties capable of reacting with twoother moieties in another molecule or two other molecules, which can bethe same or different.

[0049] The terminus typically has a branching moiety covalently linkedto a polymer chain of the polymer backbone through a hydrolyticallystable linkage. Typically, there are two free reactive groups branchingout from the branching moiety. The term “free” as used herein means thateach of the two free reactive groups has two ends, one of which iscovalently linked to the branching moiety and the other end is notlinked to any other moiety or group through covalent linkage, and(available for reaction with another moiety or group, e.g., of anothermolecule).

[0050] Typically the branching moiety is a stable, non-reactive, andinert moiety that is covalently linked to a polymer chain and to the tworeactive groups. The branching moiety should not form a hydrogen bond orionic bond with metal ions or moieties or molecules. It is believed thatthe ability to form strong hydrogen bonds or ionic bonds would interferewith the branching moiety's function. The branching atom, i.e., the atomthe two free reactive groups are linked to is not a nitrogen atom (N),but is typically a carbon atom (C).

[0051] At least one of the two free reactive groups may comprise twoportions: a reactive moiety at the free end and a tethering grouplinking the reactive moiety to the branching moiety. The reactive moietyis a moiety capable of reacting with a moiety in another molecule, e.g.,a biologically active agent such as proteins, peptides, etc. Examples ofsuitable reactive moieties include, but are not limited to, activeesters, active carbonates, aldehydes, isocyanates, isothiocyanates,epoxides, alcohols, maleimides, vinylsulfones, hydrazides,dithiopyridines, N-succinimidyl, and iodoacetamides. The selection of afree reactive moiety is determined by the moiety in another molecule towhich the free reactive moiety is to react. For example, when the moietyin another molecule is a thiol moiety, then a vinyl sulfone moiety ispreferred for the free reactive moiety of the activated polymer. On theother hand, an N-succinimidyl moiety is preferred to react to an aminomoiety in a biologically active agent.

[0052] The tethering group can have a predetermined length such that thereactive moiety linked to it is at a predetermined distance away fromthe branching moiety, and consequently, a predetermined distance fromthe other reactive moiety of the terminus. Typically, the tetheringgroup is non-reactive and is a substantially linear chain of atoms,e.g., alkyl chains, ether chains, ester chains, amide chains, andcombinations thereof.

[0053] Thus, in a preferred embodiment, the activated polymer of thisinvention can be represented by formula I:

[0054] or formula II:

[0055] In the above formula, POLY is a linear polymer chain of a watersoluble substantially non-immunogenic polymer backbone, preferably ispoly(ethylene glycol) or a derivative thereof. In the activated polymerrepresented by formula I, the polymer backbone has only one polymerchain. Y is a hydrolytically stable linkage, which can comprise an atomor a group such as —O—, —S— and —CO—NH—. It will be apparent to skilledartisan that many other hydrolytically stable linkages can also beemployed in this embodiment.

[0056] X and X′ are free reactive groups, which can be same ordifferent, each having a reactive moiety capable of reacting with amoiety in another molecule such as a protein. In the activated polymeras represented by formula I, the polymer backbone POLY can have acapping group at the end opposite to the terminus having proximalreactive groups. The capping group can be, for example, —OH, variousalkyl, and can also contain proximal reactive groups —Y—CHXX′ wherein Y,X and X′ are as described above. Accordingly, the activated polymer canhave two terminals with proximal reactive groups, one on each end of thepolymer backbone.

[0057] In formula II, R is the central core as described above. POLY isa polymer chain of the water soluble substantially non-immunogenicpolymer backbone. Y is a hydrolytically stable linkage n is from 2 to200 representing the number of polymer chains or arms in the polymerbackbone, as described above.

[0058] As will be apparent, the branching moiety as described above isCH in this embodiment of the activated polymer. Typically it does notbecome charged in normal conditions, and does not form an ionic bondwith a metal ion.

[0059] In a preferred embodiment, X and X′ can have a tethering group inaddition to a reactive moiety and can be represented by —W-Z and —W′-Z′respectively, in which Z and Z′ represent free reactive moieties forconjugating the polymer to another molecule. W and W′ representtethering groups. Z and Z′ can be different or same reactive moieties.

[0060] Some examples of preferred embodiments of the activated polymersof this invention are provided as follows:

CH₃O—PEG-CO—NH—CH(CH₂—OCO₂—NS)₂

CH₃O—PEG-CO—NH—CH(CH₂—O₂C—CH₂CH₂—CO₂—NS)₂

CH₃O—PEG-CO—NH—CH(CH₂—CO₂—NS)₂

CH₃O—PEG-O—CH(CH₂—OCO₂—NS)₂

CH₃O—PEG-O—CH(CH₂—O₂C—CH₂CH₂—CO₂—NS)₂

(OHC—CH₂CH₂—O—CH₂)₂—CH—NHCO—PEG-CONH—CH(OCH₂CH₂—CHO)₂

PEG-[CONH—CH(OCH₂CH₂—CHO)₂]₄

CH₃O—PEG-CO—NH—CH(CO₂—NS)(CH₂CH₂CO₂—NS)

[0061] In these examples, —NS represents the N-succinimidyl moiety.

[0062] In accordance with another aspect of this invention, a method forpreparing the activated water soluble polymer of this invention is alsoprovided.

[0063] Typically, in the first step, there is a first intermediatepolymer provided having a polymer backbone and a reactive end groupcovalently linked to of the polymer backbone.

[0064] In addition, a compound having three reactive groups linked to abranching moiety is provided. This compound typically has a branchingmoiety forming a central core and three free groups branching out fromthe central core. When the three free groups are linked to the same atomin the branching moiety, the atom is not a nitrogen atom. One of thethree free groups can react with the reactive end group of the firstintermediate polymer to form a hydrolytically stable linkage. The othertwo free groups can be ultimately converted into the two free reactivegroups on the terminus of the activated polymer of this invention.Examples of these compounds include, H₂NCH(CH₂—OH)₂, NaO—CH(CH₂—O—Bz)₂,H₂N—CH(CH₂CO₂H)₂, and the like. As will be apparent, in these examples,the branching moiety is CH. The H₂N— and NaO— moieties can be used tolink the compound to the first intermediate polymer to form ahydrolytically stable linkage, while the hydroxyl groups, carboxylicacid groups, and —CH₂—O-Bz groups can be ultimately converted into freereactive moieties of the activated polymer of this invention.

[0065] Thus, in the second step of the method, the compound having threereactive groups is reacted with the first intermediate polymer to form asecond intermediate polymer which includes a hydrolytically stablelinkage linking the first intermediate polymer and the compound havingthree reactive groups, thus leaving only two free groups at the terminusof the polymer chain.

[0066] In the third step, the two free groups of the compound areconverted into two free reactive moieties linked to the branchingmoiety. A number of methods known in the art can be employed in theconversion. For example, the free groups can be reacted to a compoundwhich can impart a free reactive moiety. Alternatively, the two freegroups in the second intermediate polymer can be oxidized or reduced orsubstituted to form two new free reactive moieties. Such methods will beapparent to skilled artisan in view of the Examples given below.

[0067] In accordance with yet another aspect of this invention, aconjugate is provided formed by covalently linking the activated watersoluble polymer of this invention to another molecule, e.g., abiologically active agent. Typically, a suitable biologically activeagent can be any biologically active agent having a moiety capable ofreacting with at least one of the two proximal reactive groups in theterminus of the activated polymer.

[0068] The biologically active agent can have two such moieties and eachof them can be linked to one of the two reactive groups. Alternatively,the conjugate can have two biologically active agents each being linkedto one of the two reactive moieties of the activated polymer. Forexample, the reactive moieties in the activated polymer can be vinylsulfone moieties, which can react with a thiol moiety. If a protein hasonly one thiol moiety, then two of such protein molecules can be linkedto the activated polymer through the two vinyl sulfone moieties. When aprotein has two thiol moieties, the reaction between the protein and theactivated polymer can be controlled such that each activated polymermolecule is conjugated to two protein molecules. Alternatively, thereaction can also be controlled such that the two vinyl sulfone moietiesof an activated polymer are reacted with two thiol moieties on the sameprotein molecule.

[0069] Other moieties in biologically active agents useful for reactingwith the free reactive moieties of the bivalent terminus of an activatedpolymer of this invention include, e.g., amino groups, carboxylic acidgroups, etc. It will be apparent for skilled artisan once apprised ofthe present invention to select appropriate free reactive moieties in anactivated polymer for reaction with a given moiety in a biologicallyactive agent. For example, if conjugation is through reaction with anamino group in a biologically active agent, moieties such as —CO₂—NS oraldehyde is preferably used as a free reactive moiety in the activatedpolymer for conjugation.

[0070] Because activated polymers having different tethering groups canbe prepared in accordance with this invention, an activated polymer canbe provided in which the two reactive groups in a bivalent terminus ofthe activated polymer are in a desirable distance from each other. Whensuch an activated polymer is conjugated to two biologically active agentmolecules, the two molecules can be held at a desired distance apart.

[0071] The following examples are given to illustrate the invention, butshould not be considered in limitation of the invention:

EXAMPLE 1

[0072] Synthesis of mPEG_(20K)-OCH₂CH₂CONHCH (CH₂O₂CCH₂CH₂CO₂NS)₂(NS=N-succinimidyl)

EXAMPLE 2

[0073] Synthesis of mPEG_(20K)-OCH(CH₂—SO₂CH═CH₂)₂

EXAMPLE 3

[0074] Synthesis of mPEG_(5K)-O₂CNH—CH(CH₂CO₂NS)₂

EXAMPLE 4

[0075] Synthesis of mPEG_(5k)-O—CH₂CH₂CH(CO₂H)₂

EXAMPLE 1

[0076] Reactions:

mPEG_(20K)-OCH₂CH₂CO₂NS+H₂NCH(CH₂—OH)₂→mPEG_(20K)-OCH₂CH₂CONHCH(CH₂OH)₂+NHS  1

[0077] NS=N-succinimidyl;

[0078] NHS=N-hydroxysuccinimide

MPEG_(20K)-OCH₂CH₂CONHCH(CH₂—OH)₂+2SA→mPEG_(20K)-OCH₂CH₂CONHCH(CH₂—O₂CCH₂CH₂CO₂H)₂  2

[0079] SA=succinic anhydride

mPEG_(20K)-OCH₂CH₂CONHCH(CH₂—O₂CCH₂CH₂CO₂H)₂+NHS+DCC→MPEG_(20K)-OCH₂CH₂CONHCH(CH₂—O₂CCH₂CH₂CONS)₂  3

[0080] DCC=dicyclohexylcarbodiimide

[0081] Preparations:

[0082] 1. mPEG_(20K)-OCH₂CH₂CONHCH(CH₂OH)₂

[0083] A solution of mPEG_(20K)-OCH₂CH₂CO₂NS (mSPA 20K, 20 g, 0.001moles)), H₂NCH(CH₂—OH)₂ (serinol,, 0.14 g, 0.00154 moles), andtriethylamine (0.3 ml) in acetonitrile (100 ml) was stirred undernitrogen overnight and the solvent removed by distillation. The productwas chromatographed on DEAE sepharose eluted with water and the eluatewas saturated with NaCl and extracted with chloroform. The resultingchloroform phase was dried over magnesium sulfate, filtered, and thefiltrate evaporated to dryness under vacuum to yield 20 g of product asa white solid showing a single peak with gel permeation chromatography(Ultrahydrogel 250, pH 7.2 buffer).

[0084] 2. mPEG_(20K)-OCH₂CH₂CONHCH(CH₂—O₂CCH₂CH₂CO₂H)₂

[0085] A solution of the product from (1) (20 g, 0.002 moles) andbutylated hydroxytoluene (BHT) (0.02 g) in 220 ml of chloroform wassubjected to distillation until about 150 ml of solvent had distilled.Succinic anhydride ( 2.0 g, 0.02 moles), pyridine (1.62 ml, 0.02 moles),and 40 ml of toluene were added and the resulting mixture heated at 84□C. for 20 h under nitrogen. The product was precipitated with 850 ml ofether and collected by filtration. After drying, the product wasdissolved in 200 ml of water, 20 g of NaCl added, and the pH adjusted to3 with aqueous phosphoric acid. The product was extracted withchloroform (200+150+100 ml) and the combined extracts dried overmagnesium sulfate. Evaporation of the dried solution yielded the productas a white solid (16 g). The molecular weight was determined to be20,940 Da by potentiometric titration.

[0086] 3. mPEG_(20K)-OCH₂CH₂CONHCH(CH₂—O₂CCH₂CH₂CONS)₂

[0087] A solution of the product from (2) (15 g, 0.0015 moles),N-hydroxysuccinimide (0.21 g, 0.00179 moles), dicyclohexylcarbodiimide,0.37 g, 0.00177 moles) in methylene chloride (100 ml) was stirred atroom temperature under nitrogen overnight. The suspension was filtered,product precipitated twice from methylene chloride by addition of ether(850 ml) and collected by filtration to obtain a white solid (13.0 g)which had 97.7% substitution by proton nmr. The proton nmr displayed abroad multiplet at 3.50 ppm (PEG backbone methylene groups), a singletat 3.23 ppm PEG methyl), a singlet at 2.80 ppm (NS methylenes), andmultiplets at 2.68 and 2.95 ppm ( succinate methylenes).

EXAMPLE 2

[0088] Reactions:

HO—CH(CH₂—O-Bz)₂+NaH(toluene)→NaO—CH(CH₂OBz)₂   1

[0089] Bz=Benzyl

NaO—CH(CH₂—O-Bz)₂+mPEG_(20K)-O-Ms→mPEG20KO—CH(CH₂OBz)₂   2

[0090] Ms=mesylate

mPEG_(20K)O—CH(CH₂OBz)2+HCO₂H/MeOH/H₂O/Pd/C→mPEG_(20K)O—CH(CH₂OH)₂   3

mPEG_(20K)O—CH(CH₂OH)₂+MsCl/Et₃N→mPEG_(20K)O—CH(CH₂OMs)₂   4

mPEG_(20K)O—CH(CH₂OMs)₂+HSCH₂CH₂OH→mPEG_(20K)O—CH(CH₂SCH₂CH₂OH)₂   5

mPEG_(20K)O—CH(CH₂SCH₂CH₂OH)₂+H₂WO₄→mPEG_(20K)O—CH(CH₂SO₂CH₂CH₂OH)₂   6

mPEG_(20K)O—CH(CH₂SO₂CH₂CH₂OH)₂+MsCl/ET₃N mPEG_(20K)O—CH(CH₂SO₂CH═CH₂)₂  7

[0091] Preparations:

[0092] 1. mPEG_(20K)O—CH(CH₂OBz)₂

[0093] A solution of 18 g (0,0641 moles) of 1,3-dibenzyloxy-2-propanolin 80 ml of toluene was distilled until 15 ml of toluene was removed.The azeotropically dried solution was then added to a suspension of 2.56g (0.064 moles) of NaH in 80 ml of toluene and the resulting mixturestirred while heating to 37-40□ C. before filtering. The filtrate wasthen added to a solution of azeotropically-dried mPEG_(20K) mesylate inabout 350 ml of toluene and the resulting mixture was heated for 20 h at125□ C. under N₂. The product was precipitated with cold ether, wash onthe filter with hexane, and dried under vacuum to yield 70.4 g of whitesolid shown to be pure by proton nmr.

[0094] 2. mPEG_(20K)O—CH(CH₂OH)₂

[0095] To a solution of 15 g (0.00075 moles) of the product from (1) in9.2 ml of formic acid and 0.8 ml of water was added 2.0 g of Pd/C (10%)and the mixture was stirred for 2 h under nitrogen. The mixture was thenfiltered and the pH of the filtrate adjusted to 7.2. The resultingsolution was extracted with CH₂Cl₂ and the extract dried over MgSO₄.Evaporation of the solution yielded 12.9 g of product which displayed nobenzyl groups in the proton nmr.

[0096] 3. mPEG_(20K)O—CH(CH₂OMs)₂

[0097] To an azeotropically-dried solution of the product from (2) (8.0g, 0.000889 moles) in toluene (100 ml) containing 0.008 g of BHT wasadded a solution of mesyl chloride (0.090 ml, 0.00116 moles) andtriethylamine (0.210 ml, 0.0011 moles) in 10 ml of ET₃N and theresulting solution was stirred overnight at room temperature undernitrogen. Ethanol (1 ml) was added and 50 ml of the solvent was removedby distillation before adding 500 ml of ether to precipitate theproduct. The product was collected by filtration and dried under vacuumto yield 7.6 g of the mesylate derivative shown by nmr to be 100%substituted.

[0098] 4. mPEG_(20K)O—CH(CH₂SCH₂CH₂OH)₂

[0099] A solution of the product of (3), (7.0 g, 0.00035 moles),mercaptoethanol (0.56, 0.0080 moles) ml, NaOH (0.22 g), in toluene (30ml and ethanol (60 ml) was heated at 60□ C. for 2 h under N₂. The pH wasadjusted to 7 and the product extracted with methylene chloride (3×100ml). After drying the extract over MgSO4, the solvent was removed andthe product precipitated with 250 ml of ethyl ether. The product wascollected by filtration and dried under vacuum to get 6.6 g of whitesolid which was shown by nmr to be 97.3% substituted.

[0100] 5. mPEG_(20K)O—CH(CH₂SO₂CH₂CH₂OH)₂

[0101] A solution containing the product from (4), 6.5 g (0.00065moles), and tungstic acid (0.16 g) in water (14 ml) was prepared and thepH adjusted to 6.6. Hydrogen peroxide (30%, 0.65 ml) was added and themixture stirred at room temperature overnight. The pH was adjusted to7.5 and the mixture stirred 1 h before extracting with CH₂Cl₂ (3×30 ml).The mixture was dried over MgSO4, filtered, and the filtrateconcentrated to 25 ml. The product was precipitated with 200 ml of etherand collected by filtration to obtain 5.3 g of product after vacuumdrying. The product was shown by nmr to have 86% substitution.

[0102] 6. mPEG_(20K)O—CH(CH₂SO₂CH═CH₂)₂

[0103] A solution of the product from (5), (5.2 g, 0.00052 moles), Et₃N(0.63 ml, 0.00452 moles), BHT (0.005 g), and MsCl (0.15 ml, 0.001938moles) in CH₂Cl₂ (25 ml) was stirred at room temperature for 42 h atroom temperature. Ethanol (1 ml) was added and the mixture was stirred15 minutes. Methylene chloride (50 ml) was added and the resultingsolution was washed with aqueous IM HCl followed by 5% aqueous Na₂HPO₄.After drying over MgSO4, the solution was concentrated to 30 ml and theproduct precipitated with 300 ml of ether. The product was collected byfiltration and dried under vacuum to yield the product (4.6 g) as awhite solid. The degree of substitution was 92.5% by nmr. The ¹H nmrspectrum (dmso-d6) displayed absorptions at 3.51 ppm (PEG backbone CH₂),3.23 ppm, CH₃O), 6.2 and 7.0 ppm, m, vinyl H. Note in this example thatY═O, W═CH₂, and Z=SO₂CH═CH₂.

EXAMPLE 3

[0104] Reactions:

mPEG_(5K)BTC+H₂N—CH(CH₂CO₂H)₂→mPEG_(5K)O₂CNHCH (CH₂CO₂H)₂   1

[0105] BTC=1-benzotriazolyl carbonate

mPEG_(5K)O₂CNHCH(CH₂CO₂H)₂+NHS/DCC→MPEG_(5K)O₂CNHCH(CH₂CO₂NS)₂   2

[0106] Preparations:

[0107] 1. mPEG_(5K)O₂CNHCH(CH₂CO₂H)₂

[0108] To a solution of β-glutamic acid (0.10 g, 0.00068 moles), boricacid (0.1 g) in 10 ml of water at pH 8 was added mPEG_(5K)BTC over 15 mwhile maintaining the pH at 8.15-8.25 by addition of NaOH solution. NaCl(6 g) was added and the pH of the solution was adjusted to 2 with 10%H₃PO₄. The product was extracted into CH₂Cl₂ (100+80+50 ml) and thecombined extracts were dried over MgSO₄. The mixture was filtered andthe filtrate evaporated under vacuum to yield 7.8 g of product. Themixture was determined to be 75.5% of the MPEG glutamic acid derivativeand 24.5% MPEG. This mixture was purified by chromatography on DEAEsepharose by first eluting with water and then eluting the desiredproduct with 0.5 M NaCl. Extraction of the product from the NaClsolution (pH 2) with methylene chloride followed by drying the extractover MgSO4 and evaporation of the solvent yielded 6.1 g of materialshown to be 100% pure by GPC.

[0109] 2. MIPEG_(5K)O₂CNHCH(CH₂CO₂NS)₂

[0110] A solution of mPEG_(5K)O₂CNHCH(CH₂CO₂H)₂ (6.0 g, 0.00116 moles),NHS (0.385 g, 0.001627 moles), DCC (0.676 g, 0.00162 moles) in methylenechloride (50 ml) was stirred overnight at room temperature undernitrogen. The resulting suspension was filtered and the filtrate wasadded to 500 ml of cold ethyl ether. The precipitate was collected byfiltration and dried under vacuum to obtain 5.5 g of product which wasshown by nmr to have 100% substitution. The ¹H nmr spectrum (dmso-d6)displayed absorptions at 3.51 ppm (PEG backbone CH₂), 3.23 ppm, CH₃O),4.29 ppm (—NHCH—), 4.05 ppm (—CH₂ —O—CONH—, 3.24 ppm (CH₂ CO2NS), 2.81(NS CH₂ ).

EXAMPLE 4

[0111]

[0112] Preparations:

[0113] 1. Preparation of CH₃—O—PEG-O—CH₂CH₂CH(CO₂H)₂ (Steps 1 and 2above)

[0114] Diethyl malonate (8.8 ml) in 150 ml of dry dioxane was addeddropwise to NaH (2.4 g) in 60 ml of toluene under argon. MPEG₅₀₀₀mesylate (30 g) in 250 ml of toluene was azeotropically distilled toremove 150 ml of toluene and the residue was added to the above diethylmalonate solution. After refluxing the mixture for 3-4 hours, it wasevaporated under vacuum to dryness and dried in vacuo overnight. Thedried material was then dissolved in 200 ml of 1N NaOH, the solution wasstirred for 2 days at room temperature, and the pH adjusted to 3 with INHCl. NaCl was added to the solution to a concentration of about 15% andthe mixture was then extracted with 350 ml of CH₂Cl₂ in severalportions. The combined extracts were dried over Na₂SO₄, concentratedunder vacuum and the product precipitated by addition ofisopropanol/ether (1:1). The product was collected by filtration anddried under vacuum overnight to obtain 24.7 g of product as a whitepowder. GPC (Ultrahydrogel 250) showed the product to be 98% pure.

[0115]¹H NMR (dmso-d6, ppm): 1.96 (t, CH₂CH₂ —C); 3.51 (br m, PEG—CH₂CH₂—O—).

[0116] 2. Preparation of CH₃—O—PEG₅₀₀₀-O—CH₂CH₂CH(CH₂OH)₂

[0117] CH₃—O—PEG₅₀₀₀-O—CH₂CH₂CH(CO₂H)₂ (5 g) was dissolved in 50 ml oftoluene and 9.8 ml of LiAlH₄ (1 M in THF) was added. After stirringovernight at room temperature, the mixture was evaporated to drynessunder vacuum and 150 ml of water and 22.5 g of NaCl were added. The pHwas adjusted to 6.5 with aqueous HCl and the resulting solution wasextracted with 3×50 ml of methylene chloride. The combined extracts weredried over Na₂SO₄ and the solution was evaporated to dryness. Theproduct was precipitated with ethyl ether and collected by filtration.After chromatography on DEAE sepharose, the product was 90% pure by GPC(Ultrahydrogel 250).

[0118]¹H NMR (dmso-d6, ppm): 3.51 (br m, PEG —CH₂CH₂—O—); 1.5 (br mult,CH; 4.32 (t, OH).

What is claimed is:
 1. A conjugate comprising: a biologically activeagent; and an activated, water soluble polymer comprising a polymerbackbone having at least one terminus bonded to a branching moietythrough a hydrolytically stable linkage, the branching moiety beingcovalently bonded to at least two reactive groups, each reactive groupcomprising a reactive moiety, said biologically active agent beingcovalently linked to at least one of said reactive groups.
 2. Theconjugate of claim 1, wherein said biologically active agent is selectedfrom the group consisting of proteins, peptides, lipids,polysaccharides, nucleotides, and derivatives thereof.
 3. The conjugateof claim 1, wherein said biologically active agent has a fr&e amine orthiol group and at least one of said reactive groups comprises anamine-reactive or thiol-reactive moiety.
 4. The conjugate of claim 1,wherein the branching moiety comprises a carbon atom.
 5. The conjugateof claim 1, wherein said terminus of the backbone is bonded to thestructure:

wherein Y is a hydrolytically stable linkage, A is a branching moiety,and X and X′, which may be the same or different, are reactive groupscomprising a reactive moiety.
 6. The conjugate of claim 5, wherein A isCH.
 7. The conjugate of claim 5, wherein at least one of said X and X′has the structure —W-Z, wherein W is a tethering group and Z is saidreactive moiety.
 8. The conjugate of claim 7, wherein W is selected fromthe group consisting of alkyl chains, ether chains, ester chains, amidechains, and combinations thereof.
 9. The conjugate of claim 7, wherein Wis selected from the group consisting of —(CH₂)_(m)—, —(CH₂)_(m)—O—,—O—(CH₂)_(m)—, —(CH₂)_(m)—O₂C—CH₂CH₂—, and —(CH₂)_(m)—O—(CH₂)_(r)—,wherein m and r are independently 1-10.
 10. The conjugate of claim 7,wherein Z is selected from the group consisting of active esters, activecarbonates, aldehydes, isocyanates, isothiocyanates, epoxides, alcohols,maleimides, vinylsulfones, hydrazides, dithiopyridines, andiodoacetamides.
 11. The conjugate of claim 7, wherein Z is selected fromthe group consisting of —OH, —CO₂H, —CHO, —SO₂—CH═CH₂, and —CO₂-Q,wherein Q is N-succinimidyl, sulfo-N-succinimidyl, or 1-benzotriazolyl.12. The conjugate of claim 1, wherein said reactive moiety is selectedfrom the group consisting of active esters, active carbonates,aldehydes, isocyanates, isothiocyanates, epoxides, alcohols, maleimides,vinylsulfones, hydrazides, dithiopyridines, and iodoacetamides.
 13. Theconjugate of claim 1, wherein said reactive moiety is selected from thegroup consisting of —OH, —CO₂H, —CHO, —SO₂—CH═CH₂, and —CO₂-Q, wherein Qis N-succinimidyl, sulfo-N-succinimidyl, or 1-benzotriazolyl.
 14. Theconjugate of claim 1, wherein said polymer backbone is selected from thegroup consisting of linear or branched poly(alkylene oxide), linear orbranched poly(vinyl pyrrolidone), linear or branched poly(vinylalcohol), linear or branched polyoxazoline, and linear or branchedpoly(acryloylmorpholine).
 15. The conjugate of claim 1, wherein saidpolymer backbone has a molecular weight of about 100 to about 100,000Da.
 16. The conjugate of claim 15, wherein said polymer backbone has amolecular weight of about 6,000 to about 80,000 Da.
 17. The conjugate ofclaim 1, wherein said polymer backbone is a poly(alkylene oxide)selected from the group consisting of poly(ethylene glycol),poly(propylene glycol), and copolymers of ethylene glycol and propyleneglycol.
 18. The conjugate of claim 1, wherein said polymer backbone ispoly(ethylene glycol) having a molecular weight of about 200 to about100,000 Da.
 19. The conjugate of claim 1, wherein the hydrolyticallystable linkage is selected from the group consisting of —O—, —S— and—CO—NH—.
 20. The conjugate of claim 1, wherein the at least two reactivegroups have the structure —W-Z and —W′-Z′, respectively, wherein Z andZ′ are said reactive moieties, and W and W′ are tethering groups. 21.The conjugate of claim 20, wherein W and W′ are selected from the groupconsisting of alkyl chains, ether chains, ester chains, amide chains,and combinations thereof.
 22. The conjugate of claim 20, wherein W andW′ are independently selected from the group consisting of —(CH₂)_(m)—,—(CH₂)_(m)—O—, —O—(CH₂)_(m)—, —(CH₂)_(m)—O₂C—CH₂CH₂—, and—(CH₂)_(m)—O—(CH₂)_(r)—, wherein m and r are independently 1-10.
 23. Theconjugate of claim 20, wherein Z and Z′ are each independently selectedfrom the group consisting of active esters, active carbonates,aldehydes, isocyanates, isothiocyanates, epoxides, alcohols, maleimides,vinylsulfones, hydrazides, dithiopyridines, and iodoacetamides.
 24. Theconjugate of claim 20, wherein Z and Z′ are each independently selectedfrom the group consisting of —OH, —CO₂H, —CHO, —SO₂—CH═CH₂, and —CO₂-Q,wherein Q is N-succinimidyl, sulfo-N-succinimidyl, or 1-benzotriazolyl.25. The conjugate of claim 1, wherein said polymer has the structure:

wherein R is a central core; POLY is a polymer backbone; A is abranching atom; Y is a hydrolytically stable linkage; n is from 2 to200; and X and X′ are reactive groups comprising a reactive moiety. 26.The conjugate of claim 25, wherein at least one of said X and X′ has thestructure —W-Z, wherein W is a tethering group and Z is said reactivemoiety.
 27. The conjugate of claim 26, wherein W is selected from thegroup consisting of alkyl chains, ether chains, ester chains, amidechains, and combinations thereof.
 28. The conjugate of claim 26, whereinW is selected from the group consisting of —(CH₂)_(m)—, —(CH₂)_(m)—O—,—O—(CH₂)_(m)—, —(CH₂)_(m)—O₂C—CH₂CH₂—, and —(CH₂)_(m)—O—(CH₂)_(r)—,wherein m and r are independently 1-10.
 29. The conjugate of claim 26,wherein Z is selected from the group consisting of active esters, activecarbonates, aldehydes, isocyanates, isothiocyanates, epoxides, alcohols,maleimides, vinylsulfones, hydrazides, dithiopyridines, andiodoacetamides.
 30. The conjugate of claim 26, wherein Z is selectedfrom the group consisting of —OH, —CO₂H, —CHO, —SO₂—CH═CH₂, and —CO₂-Q,wherein Q is N-succinimidyl, sulfo-N-succinimidyl, or 1-benzotriazolyl.31. The conjugate of claim 25, wherein each POLY is selected from thegroup consisting of linear or branched poly(alkylene oxide), linear orbranched poly(vinyl pyrrolidone), linear or branched poly(vinylalcohol), linear or branched polyoxazoline, and linear or branchedpoly(acryloylmorpholine).
 32. The conjugate of claim 25, wherein eachPOLY has a molecular weight of about 100 to about 100,000 Da.
 33. Theconjugate of claim 32, wherein each POLY has a molecular weight of about6,000 to about 80,000 Da.
 34. The conjugate of claim 25, wherein eachPOLY is a poly(alkylene oxide) selected from the group consisting ofpoly(ethylene glycol), poly(propylene glycol), and copolymers ofethylene glycol and propylene glycol.
 35. The conjugate of claim 34,wherein each POLY is poly(ethylene glycol) having a molecular weight ofabout 200 to about 100,000 Da.
 36. The conjugate of claim 25, wherein Ris a moiety selected from the group consisting of lysine, glycerol,pentaerythritol, and sorbitol.
 37. The conjugate of claim 25, whereineach Y is selected from the group consisting of —O—, —S— and —CO—NH—.38. The conjugate of claim 25, wherein X and X′ are —W-Z and —W′-Z′,respectively, wherein Z and Z′ are said reactive moieties, and W and W′are tethering groups.
 39. The conjugate of claim 38, wherein W and W′are selected from the group consisting of alkyl chains, ether chains,ester chains, amide chains, and combinations thereof.
 40. The conjugateof claim 38, wherein W and W′ are independently selected from the groupconsisting of —(CH₂)_(m)—, —(CH₂)_(m)—O—, —O—(CH₂)_(m)—,—(CH₂)_(m)—O₂C—CH₂CH₂—, and —(CH₂)_(m)—O—(CH₂)_(r)—, wherein m and r areindependently 1-10.
 41. The conjugate of claim 38, wherein Z and Z′ areeach independently selected from the group consisting of active esters,active carbonates, aldehydes, isocyanates, isothiocyanates, epoxides,alcohols, maleimides, vinylsulfones, hydrazides, dithiopyridines, andiodoacetamides.
 42. The conjugate of claim 38, wherein Z and Z′ are eachindependently selected from the group consisting of —OH, —CO₂H, —CHO,—SO₂—CH═CH₂, and —CO₂-Q, wherein Q is N-succinimidyl,sulfo-N-succinimidyl, or 1-benzotriazolyl.
 43. The conjugate of claim 1,wherein the polymer has a formula selected from the group consisting of:CH₃O—PEG-CO—NH—CH(CH₂—OCO₂—NS)₂,CH₃O—PEG-CO—NH—CH(CH₂—O₂C—CH₂CH₂—CO₂—NS)₂,CH₃O—PEG-CO—NH—CH(CH₂—CO₂—NS)₂, CH₃O—PEG-O—CH(CH₂—OCO₂—NS)₂,CH₃O—PEG-O—CH(CH₂—O₂C—CH₂CH₂—CO₂—NS)₂,(OHC—CH₂CH₂—O—CH₂)₂—CH—NHCO—PEG-CONH—CH(OCH₂CH₂—CHO)₂,PEG-[CONH—CH(OCH₂CH₂—CHO)₂]₄, CH₃O—PEG-CO—NH—CH(CO₂—NS)[CH₂CH₂CO₂—NS],CH₃O—PEG-O—CH₂CH₂CH(CO₂H)₂, CH₃O—PEG-O—CH₂CH₂CH(CH₂OH)₂, andCH₃O—PEG-O₂CNHCH(CH₂CO₂H)₂, wherein NS is N-succinimidyl.
 44. Theconjugate of claim 1, wherein the polymer backbone is branchedpoly(ethylene glycol) and the reactive groups are selected from thegroup consisting of active esters, active carbonates, aldehydes,isocyanates, isothiocyanates, epoxides, alcohols, maleimides,vinylsulfones, hydrazides, dithiopyridines, and iodoacetamides.
 45. Theconjugate of claim 1, wherein the polymer backbone comprises methoxypoly(ethylene glycol) disubstituted lysine.
 46. The conjugate of claim1, wherein the polymer backbone has the structure:

wherein poly_(a) and poly_(b) are water-soluble and non-peptidic polymerbackbones; R′ is a nonreactive moiety; and P and Q are nonreactivelinkages.
 47. The conjugate of claim 46, wherein poly_(a) and poly_(b)are both methoxy poly(ethylene glycol).
 48. The conjugate of claim 46,wherein R′ is H, methyl or a water-soluble and non-peptidic polymerbackbone.
 49. The conjugate of claim 46, wherein the polymer backbone isbonded to the structure:

wherein Y is a hydrolytically stable linkage, A is a branching moiety,and X and X′, which may be the same or different, are reactive groupscomprising a reactive moiety.
 50. The conjugate of claim 49, wherein Ais CH.
 51. The conjugate of claim 49, wherein at least one of said X andX′ has the structure —W-Z, wherein W is a tethering group and Z is saidreactive moiety.
 52. The conjugate of claim 51, wherein X and X′ havethe structure —W-Z and —W′-Z′, respectively, wherein Z and Z′ are saidreactive moieties, and W and W′ are tethering groups.
 53. The conjugateof claim 52, wherein Z and Z′ are each independently selected from thegroup consisting of active esters, active carbonates, aldehydes,isocyanates, isothiocyanates, epoxides, alcohols, maleimides,vinylsulfones, hydrazides, dithiopyridines, and iodoacetamides.